Proteins must organize into complexes that assemble and disassemble depending on cellular needs, while simultaneously avoiding catastrophic aggregation. Such aggregation is critical for important human diseases, such as Alzheimer's, Parkinson's, and Huntington's diseases, and perhaps even aging itself. This competitive renewal builds from an observation made in the first funding cycle of grant GM076536 (Cell chips for genome- wide protein and RNA localization in single cells): hundreds of distinct, normally cytosolic protein species form metabolite-induced, reversible, macroscopic punctate foci in quiescent cells of the yeast S. cerevisae. Are these macroscopic assemblies functional or catastrophic? The answers to this question are important for our understanding of quiescent cells and of aggregation-based disease. If these assemblies are functional or contain functional proteins, this represents unexpectedly high levels of organization in quiescent cells, with important ramifications for human cells, which are in the quiescent state for the majority of their lives. If these assemblies represent widespread aggregation, such structures are likely important factors for aging and aggregation-related disease. More generally, might protein aggregation be a novel regulatory mechanism? This grant proposes mass spectrometry proteomics, genetics, cell biology, and imaging experiments to evaluate these hypotheses and to better understand the mechanistic basis for this widespread, reversible protein aggregation. Experiments in Specific Aim I focus on determining kinetics, regulation, and mechanisms of foci formation in yeast cells. Only rarely is there a basic form of regulation in yeast that doesn't exist in humans, and initial tests suggest these foci also form in human cells. Thus, in Aim II, we aim to determine more broadly if these foci, and their regulatory logic, are conserved in human cells and hence likely selected for evolutionarily. Lastly, initial evidence suggests foci formation increases both in replicatively and chronologically aged cells. Experiments in Aim III will define the contribution of foci formation and aggregation towards cellular lifespan, and determine the causal relationship between aggregation and aging. These will provide concrete evidence, on a protein-by-protein basis, either for forming functional protein assemblies in quiescent cells or for catastrophic aggregation. This work will define the extent to which protein foci and aggregates govern survival in quiescent cells and impact cellular lifespan, and is a step towards better understanding of the forces underlying aggregation diseases and aging.